This research program is concerned with the analysis, synthesis, protein expression, and structure-function relationships of peptide and protein hormones. Current emphases are placed on the structure and function of the angiotensin II (Ang II) and gonadotropin-releasing hormone (GnRH) receptors. Recent accomplishments of these projects include milligram scale synthesis and purification of a sulforhodamine 101 (Texas Red) conjugate of [D-Lys6, des Gly10amide]GnRHethylamide at epsilon-amino group of D-Lys6. The reaction of [D-Lys6, des Gly10amide]GnRHethylamide with N-succinimidyl ester of sulforhodamine 101 yielded five components as revealed by reverse phase high performance liquid chromatography in which the major component was characterized as the mono-sulforhodamine 101 acylated [D-Lys6, des Gly10amide]GnRHethylamide at epsilon-amino group of D-Lys6. This major conjugate is biologically active as revealed on confocal microscopy by its ability to bind to the GnRH receptor and to internalize in the same manner as the GnRH agonist. In the area of studies on the structure and function of the Ang II receptor, the association of hAT1 and MHC class II-associated invariant chain, Ii has been demonstrated in vivo by yeast mating experiments and by co-localization at the plasma membrane of human embryonic kidney (HEK-293) cells on confocal microscopy. For co-localization studies, HEK cells were transfected with both AT1-green fluorescent protein and Ii-red fluorescent protein fusion proteins. In this cell line, inhibition of hAT1 receptor internalization in response to Ang II stimulation in the presence of Ii on the plasma membrane was found. The manner in which the Ii chain interferes with agonist-induced endocytosis of the AT1 receptor is under further investigation. We also have developed a chemical method that converts phospho-Ser and phospho-Thr to cysteic acid and beta-methyl cysteic acid, respectively for direct sequencing of the Ser and Thr phosphorylation sites by electron-spray ionization tandem mass spectrometry. Five model phosphopeptides including three synthetic P-Ser, P-Thr or both P-Ser and P-Thr containing peptides, a protein kinases C-phosphorylated bovine myelin basic protein residues 4-14 peptide, and a phosphopeptide derived from beta-casein trypsin digests were modified and then sequenced using an ESI-quadrupole ion trap mass spectrometer. Following incubation of P-Ser or P-Thr containing peptides with 0.6 M Na-sulfite in 0.1 N NaOH ay room temperature, 90% P-Ser and 80% P-Thr was converted to cysteic acid and beta-methyl cysteic acid respectively, as revealed by amino acid analysis. The conversion can be carried out at 1 micromolar concentration of the peptide. Both cysteic acid and beta-methyl cysteic acid residues in the sequence were shown to be stable and easily identifiable in a series of y and b ions under general conditions for tandem mass spectrometric sequencing applicable to common peptides.